Journal: bioRxiv

Article Title: Salivary exosomal miR-1307-5p predicts disease aggressiveness and poor prognosis in oral squamous cell carcinoma patients

doi: 10.1101/2022.07.13.499918

Figure Lengend Snippet: (a) Representative TEM images of exosomes from OSCC patients, (Scale: 200nm); (b) Estimation of size and concentration of salivary exosomes from OSCC patients using Nanoparticle Tracking Analysis (NTA). The x-axis indicates the size distribution of particles and the y-axis shows the signal intensity in NTA; (c-e) Characterization of exosomes by flow cytometry. We used a membrane-binding dye vFRed to stain exosomes and examined the violet side scatter v/s vFRed staining of these exosomes. This profile was similar to the profile of Lipo-50 liposomes used as a reference standard. Salivary exosomes were stained with antibodies against tetraspanins (CD63, CD81 and CD9) labelled with PE, and APC labelled CD47 antibody. We identified a distinct CD47+/vFRed+ population in the exosome population that was absent in the unstained control. This population was then examined for tetraspanin expression and showed detectable levels of tetraspanins. Since these particles stain with vFRed, have a violet side scatter similar to 50 nm liposomes, express CD47 and tetraspanins, we concluded that these are exosomes.

Article Snippet: The samples (at optimal dilution) were then incubated with PE tagged antibodies against tetraspanins (TS; CD9, CD63, and CD81) (Cellarcus Biosciences, CA, USA) and APC tagged CD47 antibody (Thermo Scientific, Waltham, MA, USA) for one hour at room temperature.

Techniques: Concentration Assay, Flow Cytometry, Membrane, Binding Assay, Staining, Liposomes, Control, Expressing

Journal: Journal of Extracellular Vesicles

Article Title: DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination

doi: 10.1080/20013078.2020.1746529

Figure Lengend Snippet: VEGF-C is associated with extracellular vesicles . (a) Representative immunohistochemical staining images (serial section) show expression of Lyve-1 and VEGF-C in the pancreas of Lox-Stop-Lox (LSL)- Trp53 R172 H (WT) and in the tumour of LSL-Kras G12D ; LSL-Trp53 R172 H ; Pdx1-cre (KPC) transgenic mouse. (b) Serum-free conditioned medium from MIA PaCa-2 cells was collected and fractions were isolated based on size exclusion chromatography according to the manufacturer’s protocol. Expression of VEGF-C, CD63 and HSP70 was detected in vesicle-associated fractions by Western blotting (upper). Three fractions (as indicated) were sent for NTA analysis (bottom left). Protein concentration in each fraction was measured (bottom right). (c) VEGF-C is highly expressed in EV fraction. Conditioned medium from MIA PaCa-2 cells was collected and ultracentrifugation was performed to isolate microvesicles (MV), exosomes (Ex) and supernatant (Sup). Western blotting was performed to detect the expression of VEGF-C, CD63 and ALBUMIN (ALB) in whole cell lysate (WCL), MV, Ex and Sup with equal amount of protein. (d) EV was isolated by ExoQuick-TC from CM of MIA PaCa-2 cells. Equal protein amount was loaded to compare VEGF-C expression in WCL, EV and CM. CD63, TSG101 and HSP70 were used as EV markers. ALB was detected to demonstrate the purity of EV (left). EV isolated by ExoQuick-TC was sent for NTA analysis (right). (e) Representative transmission electron microscopic images show that VEGF-C is associated with EV. VEGF-C (dark particles) was stained with gold particle-labelled anti-VEGF-C antibody. (f) VEGF-C is associated with surface of EV. Isolated EV (by ExoQuick-TC) was treated with proteinase K, triton X, proteinase K plus triton X and trypsin. Western blotting was performed to detect VEGF-C and GAPDH. (g) Expression of VEGF-C in AsPC1 cells. VEGF-C cDNA was cloned into pCDH lentivirus vector under the control of CMV promoter (upper panel). Expression of secreted VEGF-C is decreased by treatment with GW4869 (40 μM). (h) VEGF-C is enriched in the small EV fraction. Conditioned medium from AsPC-VEGF-C cells was collected and the EV fraction (MV and Ex) was purified by ultracentrifugation. CD63 was detected as a marker for EV. (i) NTA analysis (left) and Western blotting (right) show particle numbers and VEGF-C expression in AsPC-Ctrl and AsPC-VEGF-C cells. EV was isolated by ExoQuick-TC. (j) LECs uptake EV from MIA PaCa-2 cells. Mia PaCa-2 cells were labelled with PKH67 and serum-free conditioned medium from PKH67 labelled Mia PaCa-2 cells were collected and isolated by ExoQuick-TC. LECs were treated EVs for 6 h and fixed for image taken. (k) Quantification of LECs proliferation treated with EV from AsPC-control and AsPC-VEGF-C cells. Ki67 staining was performed as the indicator of the proliferation of LECs. ** P < 0.01. (L) Schematic of experimental design to evaluate the effect of EV from AsPC-VEGF-C cells (left). Representative immunohistochemical images show the increase of lymphatic vessels (Lyve-1) in AsPC-1 tumours treated with EV isolated from AsPC-VEGF-C cells (by ultracentrifugation isolation).

Article Snippet: Antibodies against VEGF-C (GeneTex, GTX113574), DUSP2 (Santa Cruz, sc-32776), Phospho-p44/42 MAPK (Cell Signaling Technology #4370), p44/42 MAPK (Cell Signaling Technology #4696), CD63 (Taiclone, tcba9211), GAPDH (Genetex GTX100118), Furin (Abcam, ab183495), PC5/6 (Abcam, ab40133), PCSK7 (Abcam, ab47925), HSP70 (Santa Cruz, sc-32239), Annexin V (Santa Cruz, sc-74438), SNAP-tag (New England Biolabs, P9310 S) were used for Western blotting.

Techniques: Immunohistochemical staining, Staining, Expressing, Transgenic Assay, Isolation, Size-exclusion Chromatography, Western Blot, Protein Concentration, Transmission Assay, Clone Assay, Plasmid Preparation, Control, Purification, Marker

Journal: Journal of Extracellular Vesicles

Article Title: DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination

doi: 10.1080/20013078.2020.1746529

Figure Lengend Snippet: Inhibition of DUSP2 increases VEGF-C expression . (a) Representative pictures of DUSP2 immunostaining in pancreatic tumours. PanIN: pancreatic intraepithelial neoplasia, Ca: invasive carcinoma, arrowhead: non-tumour elements. (b) Representative pictures of DUSP2 immunostaining in KPC tumours. Arrows: infiltrating cancer cells; circles: carcinoma (Ca). (c) Representative immunohistochemical staining images show the expression of ERK1/2 phosphorylation in LSL - Trp53 R172 H (WT) pancreas and KPC tumours. (d) Representative Western blots show the expression of phosphorylated ERK (left), EV-VEGF-C (middle) in MIA PaCa-2 cells treated without (DMSO) or with ERK inhibitor, SCH 772984 (1 μM, SCH) for 24 h. EVs were isolated by ultracentrifugation (Ultra) or ExoQuick-TC (ExoQ) exosome precipitation reagent. The right panel shows EV-VEGF-C in AsPC-1-Ctrl and AsPC-1-VEGF-C cells treated with DMSO or SCH 772984 (1 μM, SCH). EV in AsPC-1 was isolated by ExoQuick-TC. Annexin V and CD63 were used as markers of EVs in MIA PaCa-2 and AsPC-1 cells, respectively. (e) Representative image shows levels of DUSP2 in human pancreatic cancer cell lines PANC-1 and MIA PaCa-2 (left) and levels of DUSP2, total and phospho-ERK in control and DUSP2-KD PANC-1 cells (right). DU#1 and DU#2 indicate two stable clones of PANC-1 cells with DUSP2 knockdown. β-actin is a loading control. (f) VEGF-A and VEGF-C were measured in control and DUSP2-KD PANC-1 cells by RT-qPCR. Data represent mean and SEM of three experiments using different batches of cells (left). Representative image and the quantitative result (right) show the increase of secreted VEGF-C in DUSP2-KD PANC-1 cells. Ponceau S staining was employed to examine the equal loading of proteins. (g) RT-PCR and Western blotting for the detection of VEGF-C mRNA and secreted protein in control and DUSP2-KD PANC-1 cells treated with U0126 (10 μM). (h) Representative Western image shows levels of DUSP2 and VEGF-C in cells with or without transient expression of DUSP2 in MIA PaCa-2 cells. Secreted VEGF-C was quantified in control and DUSP2 transfected MIA PaCa-2 cells (right). (i) Inverse expression of DUSP2 and VEGF-C in mouse tissues. Various tissues from male BALB/c mice were collected and homogenized. Western blotting was performed to measure the expression of DUSP2 and VEGF-C. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies against VEGF-C (GeneTex, GTX113574), DUSP2 (Santa Cruz, sc-32776), Phospho-p44/42 MAPK (Cell Signaling Technology #4370), p44/42 MAPK (Cell Signaling Technology #4696), CD63 (Taiclone, tcba9211), GAPDH (Genetex GTX100118), Furin (Abcam, ab183495), PC5/6 (Abcam, ab40133), PCSK7 (Abcam, ab47925), HSP70 (Santa Cruz, sc-32239), Annexin V (Santa Cruz, sc-74438), SNAP-tag (New England Biolabs, P9310 S) were used for Western blotting.

Techniques: Inhibition, Expressing, Immunostaining, Immunohistochemical staining, Staining, Phospho-proteomics, Western Blot, Isolation, Control, Clone Assay, Knockdown, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Journal of Extracellular Vesicles

Article Title: DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination

doi: 10.1080/20013078.2020.1746529

Figure Lengend Snippet: DUSP2 knockdown promotes lymphangiogenesis and lymphovascular invasion in the orthotopic pancreatic cancer mouse model . (a) Conditioned media from DUSP2-KD PANC-1 cells enhanced the proliferation of LECs. Conditioned media from control and DUSP2-KD PANC-1 cells were used to treat LECs with or without the addition of VEGFR2/R3 inhibitor, Lenvatinib (10 nM). Data represent mean and SEM of three independent experiments using conditioned media collected from different batches of cells. (b) Representative pictures (left) and quantitative result (right, n = 3) show conditioned media from DUSP2-KD PANC-1 cells promoted migration of LECs. * P < 0.05; *** P < 0.001. (c) Representative picture of tumours developed in the pancreas of mice when injected with PANC-1 control and DUSP2-KD cells. (d) Representative images show the increase of lymphatic vessels in DUSP2-KD orthotopic injected tumours compared with that in control tumours. Lymphatic marker Lyve-1 was used to identify lymphatic vessels. CD31 was used to identify blood vessels. Original magnification, ×200. (e) Example of lymphovascular invasion and lymph node metastasis by H&E stain. No tumour cells were present in the lymphatic/blood vessels in mice injected with control PANC-1 cells while tumour emboli were seen in mice injected with DUSP2-KD PANC-1 cells. Asterisk represents tumour cells. Fisher’s exact test (two sided) indicates a significant increase in the number of mice with lymphovascular invasion (LVI) and nodal metastasis (LNM) if they are bearing DUSP2-KD tumours ( P = 0.01). Tumours were sliced as serial sections and H&E-stained histologic sections were scored in a blinded manner by a pathologist (bottom right). (f) Schematic drawings illustrate the strategy of generating conditional knockout of Dusp2 in the pancreas of mice (top). Representative image of genotyping result by PCR (middle). Representative immunohistochemical staining images show expression of Lyve-1 in the pancreas of 4 months old wild type (WT) and Dusp2 knockout (Dusp2 −/- ) mice (bottom). Original magnification, ×200. (g) Representative immunohistochemical staining images show expression of phosphorylated ERK, Lyve-1 and CD31 in serial sections in the pancreas of 7 months old wild type (WT) and LSL-Kras G12D ; LSL-Dusp2; Pdx1-cre (KDC) mice. Original magnification, ×200. (h) The expression of VEGF-C is increased in EV isolated from serum in KDC mice. Western blotting was performed for the detection of VEGF-C and CD63. Ponceau S staining was used as control for equal amount of protein loading.

Article Snippet: Antibodies against VEGF-C (GeneTex, GTX113574), DUSP2 (Santa Cruz, sc-32776), Phospho-p44/42 MAPK (Cell Signaling Technology #4370), p44/42 MAPK (Cell Signaling Technology #4696), CD63 (Taiclone, tcba9211), GAPDH (Genetex GTX100118), Furin (Abcam, ab183495), PC5/6 (Abcam, ab40133), PCSK7 (Abcam, ab47925), HSP70 (Santa Cruz, sc-32239), Annexin V (Santa Cruz, sc-74438), SNAP-tag (New England Biolabs, P9310 S) were used for Western blotting.

Techniques: Knockdown, Control, Migration, Injection, Marker, Staining, Knock-Out, Immunohistochemical staining, Expressing, Isolation, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination

doi: 10.1080/20013078.2020.1746529

Figure Lengend Snippet: DUSP2 regulates the secretion of EV-VEGF-C in pancreatic cancer cells . (a) Representative (upper) and quantification (bottom) of VEGF-C and HSP70 expression by Western blotting in EV (isolated by ExoQuick-TC) from control and DUSP2-KD PANC-1 cells. (b) Representative Western blots show VEGF-C expression in WCL, EV and CM in DUSP2-KD PANC-1 cells (left). Representative Western blots show VEGF-C expression in EV isolated from DUSP2-KD PANC-1 cells treated with DMSO (control) and SCH 772984 (1uM) for 24 h (right). CD63 and HSP70 were detected as EV markers. ALB was detected to demonstrate the purity of EV. Equal amount of protein (20 ug) was loaded. EV was isolated by ExoQuick-TC. (c) Images of control and DUSP2-KD PANC-1 cells transfected with VEGF-C-SNAP tag for 24 h. Cells were treated with proprotein convertase inhibitor in serum-free medium for another 24 h after transfection. After labelling by the fluorescent substrate for SNAP, cells were fixed and imaged. (d) Images of control and DUSP2-KD PANC-1 cells taken by the transmission electron microscope. Red arrows indicate positive staining by 10 nm gold labelled anti-VEGF-C antibody. (e) Nanoparticle Tracking Analysis (NTA) was performed to detect secreted particles from control and DUSP2-KD cells. Serum-free conditioned medium was collected and centrifuged to remove debris and were sent for NTA. The X-axis represents the size of particles and the Y-axis represents the number of particles secreted per cell. DUSP2-KD cells have increased numbers of EV ranging from 85 to 155 nm (middle panel). EV secreted from DUSP2-KD cells was diminished if treated with GW4869 (20 μM) (right panel). (f) Inverted light sheet microscope (Luxengo) was used to track fast-moving particles in control and DUSP2-KD cells. Cells were labelled with PKH67 and plated into the cell holder for the tracking of PKH67 positive particles within the cells. 3D tracking of PKH67 particles in control and DUSP2-KD cells analysed by Imaris software (left). Particles in DUSP2-KD cells have increased speed (upper right) and track length (bottom right). Experiments have been performed two times and represented data are shown. (g) DUSP2-KD cells have an increased rate of EV-VEGF-C secretion. VE-Snaptag was stably expressed in control and DUSP2-KD cells. Cells were treated with proprotein convertase inhibitor (20 uM) in serum-free RPMI. EV was isolated from the medium at a different time point. Western blotting was performed to detect unprocessed VEGF-C (VE-Snap) in EV (upper). Expression of EV-VEGF-C in control and DUSP2-KD cells by fold change analysis over 2 h (lower). (h) Schematic of experimental design to investigate the function of EV from DUSP2-KD PANC-1 cells (left). Representative immunohistochemistry images show the increase of lymphatic vessels (Lyve-1) in PANC-1 tumours treated with EV isolated from DUSP2-KD cells.

Article Snippet: Antibodies against VEGF-C (GeneTex, GTX113574), DUSP2 (Santa Cruz, sc-32776), Phospho-p44/42 MAPK (Cell Signaling Technology #4370), p44/42 MAPK (Cell Signaling Technology #4696), CD63 (Taiclone, tcba9211), GAPDH (Genetex GTX100118), Furin (Abcam, ab183495), PC5/6 (Abcam, ab40133), PCSK7 (Abcam, ab47925), HSP70 (Santa Cruz, sc-32239), Annexin V (Santa Cruz, sc-74438), SNAP-tag (New England Biolabs, P9310 S) were used for Western blotting.

Techniques: Expressing, Western Blot, Isolation, Control, Transfection, Transmission Assay, Microscopy, Staining, Software, Stable Transfection, Immunohistochemistry

Journal: PLoS ONE

Article Title: Commercial Cow Milk Contains Physically Stable Extracellular Vesicles Expressing Immunoregulatory TGF-β

doi: 10.1371/journal.pone.0121123

Figure Lengend Snippet: (A) Size distribution of isolated EVs observed in a NanoSight LM12. Raw data was analyzed with NTA software, with a minimum expected particle size of 50nm. At least 200 tracks had to be analyzed per sample for inclusion in final analysis. Data presented is a representation of >15 samples in multiple experiments. (B) Electron microscopy of the ultracentrifugation pellet from milk showed both exosomes with spherical shapes (30–100nm) and larger EVs ranging from 100–200nm. (C) Exosome capture assay, exosomes were captured with an anti-CD63 antibody, prior to elution and validation by NTA. (D) Detection of bovine specific RNA in EVs. β -Casein, β-Lactoglobulin (β LG) and elongation factor-1α (EF1α) were detected by RT-qPCR in both colostrum and commercial milk. (E) Detection of immunoregulatory miRNAs in EVs, including miR-21, miR-30a, miR-92a, miR-99a and miR-223.

Article Snippet: Antibodies against bovine-CD63 (1μg/ml) (MCA2042G, Bioconnect, Huissen, The Netherlands) were coated onto Maxisorp Nunc-Immuno plates (Thermo Scientific, Roskilde, Denmark) overnight at 4°C.

Techniques: Isolation, Software, Electron Microscopy, Biomarker Discovery, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Digital Detection of Exosomes by Interferometric Imaging

doi: 10.1038/srep37246

Figure Lengend Snippet: The expression of the typical exosomal biomarkers CD81 CD63 as well as the neural adhesion protein CD171 is significantly different than in the artificial CSF, negative control. An additional negative control, a non correlated IgG, shows a low level of non-specific binding. Each multiplexed chip was run in duplicate independent replicate tests.

Article Snippet: Monoclonal antibodies against the human tetraspanins CD63, CD81, CD9, and CD171 were purchased from Nexgenarrays LLC (Boston, MA).

Techniques: Expressing, Negative Control, Binding Assay